Prediction of network pharmacology, molecular docking-based strategy, and vitro assays to determine potential pharmacological mechanism of Dioscoreae bulbiferae and Bruceae fructus against laryngocarcinoma

Background: Based on network pharmacology, molecular docking, and vitro assays, investigate the probable pharmacological mechanism of Dioscoreae bulbiferae and Bruceae fructus in the treatment of laryngocarcinoma. Methods: The active components and targets of Dioscoreae bulbiferae and Bruceae fructus were retrieved from the Traditional Chinese Medicine Systems Pharmacology Database and Analysis Platform database. Targets linked with laryngocarcinoma were gathered from the GeneCards, DisGeNET, and DrugBank databases. The String database was utilized to build a protein–protein interaction network of common medication and illness targets, after which the core targets were filtered out. The Metascape database served for gene ontology enrichment and Kyoto encyclopedia of genes and genomes pathway analysis of common targets. AutoDock then performed molecular docking between the essential component and the vital target. To investigate the biological effects of diosbulbin B, we assessed the viability of laryngocarcinoma cells after diosbulbin B therapy using the Mahalanobis Taguchi system technique. Following that, we looked at how diosbulbin B affected colony formation after 14 days of culture of treated cells. Flow cytometry was utilized to detect apoptosis in order to examine the influence of diosbulbin B on laryngocarcinoma cell apoptosis. Results: According to a study of the literature, the fundamental components of Dioscoreae bulbiferae and Bruceae fructus in the treatment of laryngocarcinoma include brusatol and diosbulbin B, which may operate on core targets such as cyclin D1, Cyclin Dependent Kinase Inhibitor 1A, and E2F Transcription Factor 1. The significant pathways discovered using Kyoto encyclopedia of genes and genomes enrichment analysis were the phosphoinositide 3-kinase-protein kinase B signaling route, the tumor necrosis factor signaling pathway, and so on. These pathways primarily influence the development and prognosis of laryngeal cancer by controlling cell growth, cell proliferation, angiogenesis, tumorigenesis, and metastasis. The molecular docking studies revealed that the affinity between the heart and crucial targets was robust. The results of vitro assays indicate that diosbulbin B suppressed Hep-2 cell activity in a concentration-dependent manner. Besides, diosbulbin B has powerful antiproliferative properties in Hep-2 cells. Flow cytometry results showed that diosbulbin B promoted laryngocarcinoma cell apoptosis in a concentration-dependent manner. Conclusion: The article delivered a preliminary discussion of the probable mechanism of Dioscoreae bulbiferae and Bruceae fructus in the treatment of laryngocarcinoma, which can serve as a theoretical basis and evidence for subsequent experimental investigation.


Introduction
Squamous cell carcinoma is the most prevalent kind of cancer in the larynx.Squamous cell carcinoma of the larynx accounts for 30% of all squamous cell carcinomas.Current morbidity and death rates are high. [1,2]There have been around 13,000 instances of laryngeal cancer documented in the United States.Each year in China, approximately 25,300 new cases of laryngeal cancer are recorded. [3]The leading causes of laryngeal cancer have been identified as smoking and drinking.The danger varies concerning the location of the laryngeal tumor.Alcohol and cigarette usage have a higher influence on suprapharyngeal geography.Other occupational exposure risk factors, such as coal dust, cemented carbide dust, and chlorinated solvents, have also been linked to laryngeal cancer. [4]So nevertheless, the precise mode of action of laryngeal carcinoma is unknown.Consequently, understanding the molecular etiology of laryngeal cancer growth is critical for developing novel therapeutic techniques.
Dioscoreae bulbiferae is a dried tuber of the perennial herbaceous twining vine Dioscorea zingiberensis.Produced mostly in Hubei, Hunan, Jiangsu, and other locations.In therapeutic practice, Xanthate-based formulations and ointments are commonly utilized.Xanthophyll is the principal active component in diosbulbin B. [5] Xanthophyll has been demonstrated in studies to help prevent and cure cancers such as esophageal cancer, nasopharyngeal cancer, maxillary sinus cancer, gastric cancer, and others. [6,7]Bruceae fructus is a Momordica shrub native to our country's southeast and other tropical and subtropical regions.A traditional Chinese medicine, anti-tumor injection derived from the fruit of Bruceae fructus has been widely utilized as an adjuvant treatment for lung cancer, lung cancer brain metastases, and gastrointestinal malignancies.Though Bruceae fructus is widely used as an effective injectable for anti-tumor, but it also comes in many oral forms, such as Brucea oil oral lotion.Clinical trials have demonstrated that Bruceae fructus can improve the effectiveness of treatment in patients with clinically advanced non-small cell lung cancer. [8,9]Bruceae fructus bitol is a key active component of the plant. [10]13][14] In traditional Chinese medicine, Dioscoreae bulbiferae and Bruceae fructus are often used in combination in clinical practice.Therefore, Dioscoreae bulbiferae and Bruceae fructus were chosen for this study.
Network pharmacology is an emerging interdisciplinary field that is based on systems biology theory and integrates computer science and bioinformatics.Network pharmacology can be used to analyze the "multi-component, separate-target, multipathway" synergistic relationship between drugs, diseases, and targets.It has played an instrumental part in understanding the pharmacological mechanism of traditional Chinese medicine, examining the toxicological mechanism of traditional Chinese medicine, and researching and creating novel traditional Chinese medicine. [15]Molecular docking utilizes receptors and ligands with known structures to detect interactions between molecules and anticipate the optimum binding mode between molecules using the 3 complimentary concepts of geometry, energy, and chemical environment.It offers significant value and prospective benefits in the research of the possible target and action mechanism of active components in traditional Chinese medicine, as well as the study of the pharmacological mechanism of Chinese herbal compound prescription. [16]n conclusion, Squamous cell carcinoma is the most prevalent kind of cancer in the larynx.Squamous cell carcinoma of the larynx accounts for 30% of all squamous cell carcinomas.Current morbidity and death rates are high.Dioscoreae bulbiferae and Bruceae fructus are extensively utilized in the treatment of laryngocarcinoma in China.Brucea javanica oil emulsion has obvious induction of apoptosis on human laryngeal cancer Hep-2cells in a dose-dependent and time-dependent manner.However, the fundamental mechanism of Dioscoreae bulbiferae and Bruceae fructus in the treatment of laryngocarcinoma remains unknown.Thus, based on network pharmacology and molecular docking, we perform this research to investigate the probable pharmacological mechanism of Dioscoreae bulbiferae and Bruceae fructus in the treatment of laryngocarcinoma.The graphical abstract was shown as follows (Fig. 1).

Screening of compositions and targets in Dioscoreae bulbiferae and Bruceae fructus
The chemical compositions of Dioscoreae bulbiferae and Bruceae fructus were collected from the TCMSP database, and the active ingredients were screened according to Adsorption, Distribution, Metabolism, Excretion, meeting the 2 conditions of oral bioavailability (OB) ≥ 30 % and drug-like ≥ 0.18.After the active ingredients of each drug were obtained, the corresponding targets were also searched in the TCMSP database, and the target name was standardized by the Uniprot database.Cytoscape software was used to draw the active componentstargets network diagram of Dioscoreae bulbiferae and Bruceae fructus, and the topological analysis was carried out through the network analyzer function of the software.Reviewing the extensive literature, we found components which closely related to laryngocarcinoma, and defined them as core components.

Screening of laryngocarcinoma-related targets
Laryngocarcinoma-related targets were retrieved from the GeneCards, DisGeNET, and DrugBank databases using "laryngocarcinoma" (Mesh) as the key search term.After merging the targets obtained from the above database, as well as removing duplicates, the laryngocarcinoma-related targets were obtained.

Construction of a protein-protein interaction (PPI) network between interaction targets and acquisition of core targets
The interaction targets of Dioscoreae bulbiferae and Bruceae fructus and laryngocarcinoma were obtained through Venny, and then the Venn diagram was drawn.The PPI network between interaction targets was built using the String database.The mode was "Multiple Protenin," and the organism was "Homo sapiens."The minimum required interaction score was set to "Highest confidence (0.900)," and the disconnected nodes in the network were hidden.Other parameters remained unchanged.The obtained node1, node2, and combined scores were imported into the Cytoscape software for visual analysis.The network analyzer function of the software was used for www.md-journal.comtopological analysis, and the core targets of Dioscoreae bulbiferae and Bruceae fructus in the treatment of laryngocarcinoma were obtained according to the degree value.

Gene ontology (GO) enrichment and Kyoto encyclopedia of genes and genomes (KEGG) pathway analysis
GO enrichment and KEGG pathway analysis were performed on the interaction targets of Dioscoreae bulbiferae and Bruceae fructus and laryngocarcinoma using the DAVID database.The Select Identifier was set to "OFFICIAL GENE SYMBOL" and the species and background were set to "Homo sapiens."Then the cell component, molecular function (MF), biological process (BP), and KEGG pathway analysis were performed.The data was exported and sorted according to the P value.For each GO enrichment and KEGG pathway, 20 items with the lowest P value were selected to draw the advanced bubble diagram by Bioinformatics.

Construction of targets-pathways interaction network
The 20 items with the lowest P value in the KEGG pathways were imported into Cytoscape software, and the interaction targets between the compounds and disease targets included in the above KEGG pathways were also imported into the software, then the targets-pathways interaction network was constructed.

Molecular docking
AutoDock molecular docking was performed between the selected core components and the core targets.The mol2 formats of the core components were downloaded from the TCMSP database, and transformed into pdb formats by using PyMOL, then saved as pdbqt formats by using AutoDock Tools.
The pdb formats of the core targets was downloaded from the PDB database (the protein complex with ligand and resolution of <3 A was selected), and the water removal, hydrogenation, and removal of the original ligand were performed by PyMOL.The atomic type was set as Assign AD4 type, and then imported into AutoDock Tools to save as pdbqt formats.The spatial position of the original ligand in the protein complex was defined as the active pocket, and the Lamarckian genetic algorithm was selected to run molecular docking by AutoDock.The binding free energy was used to screen the best docking results.Finally, the results were visualized by PyMOL.

Vitro assays
2.8.1.Cell lines and drugs.The Hep-2 cells were obtained from FuHeng Biology (Shanghai, China) and validated by short tandem repeat analysis before being cultivated in Dulbecco modified Eagle medium (RPMI-1640, Hyclone, USA) containing 10% fetal bovine serum (FBS; BI, Israel).In a constant temperature incubator with 5% CO 2 , all cells were incubated in a full medium.MedChemExpress (MCE, https:// www.medchemexpress.cn/bacoside-a.html,NO: HY-N0131) supplied the diosbulbin B. The diosbulbin B was kept at 4°C and dissolved in 100 mg/mL stock solution of dimethyl sulfoxide.The dimethyl sulfoxide group with a level of <1/1000 was represented by the 0 μg/mL group.

Cell viability assay. Mahalanobis Taguchi system (MTS)
tests were done on laryngocarcinoma cells using a Promega Kit (Madison, WI) according to the manufacturer's instructions.In brief, 3000 Hep-2 cells were seeded onto 96-well plates at 100 L/ well and grown under various treatment settings.Following the required time, 10 L of MTS solution was added to 90 L of RPMI-1640 each well, and the plates were incubated for 30 minutes.Following that, the absorbance of each well was measured at 490 nm using a microplate reader (Bio-Rad, USA).

Obtainment of components and targets in Dioscoreae bulbiferae and Bruceae fructus
The components and targets of Dioscoreae bulbiferae and Bruceae fructus were obtained from the TCMSP database, and OB ≥ 30% and drug-likeness ≥0.18 were used as the included criteria.After eliminating the non-target components and removing the duplication, 29 active components were obtained.Among them, there are 14 unique ingredients of Dioscoreae bulbiferae, and 14 unique ingredients of Bruceae fructus.There was 1 common ingredient of Dioscoreae bulbiferae and Bruceae fructus.The active components of Dioscoreae bulbiferae and Bruceae fructus were arranged according to the OB value from large to small, which shown in Tables 1 and 2. By retrieving the TCMSP database, a total of 207 targets of Dioscoreae bulbiferae and 87 targets of Bruceae fructus were searched.Repetitive values were removed, and the Uniprot database was used to standardize the names of targets.Then a total of 197 targets of Dioscoreae bulbiferae and Bruceae fructus were obtained.Cytoscape software was used to draw the active components-targets interaction network of Dioscoreae bulbiferae and Bruceae fructus (Fig. 2A).Reviewing the extensive literature, we found that diosbulbin B and brusatol are closely related to laryngocarcinoma, therefore, defined them as core components.

Acquisition of laryngocarcinoma-related targets
A total of 1026 laryngocarcinoma-related targets were obtained in the GeneCards database, 456 laryngocarcinoma-related targets were obtained from the DisGeNET database and 50 laryngocarcinoma-related targets were obtained in the DrugBank database.After merging the targets obtained from the 3 databases and deleting the duplicate values, 1258 laryngocarcinoma targets were obtained.

Construction of a PPI network between interaction targets and acquisition of core targets
There were 114 interaction targets of Dioscoreae bulbiferae and Bruceae fructus and laryngocarcinoma were obtained through Venny (Fig. 2B).Importing 114 interaction targets into the String database to build PPI networks (Fig. 2C) (the disconnected nodes in the network were hidden).The obtained node1, node2, and combined scores were imported into the Cytoscape software for visual analysis.The network analyzer function of the software was used for topological analysis.According to the degree value, the core targets of Dioscoreae bulbiferae and Bruceae fructus in the treatment of laryngocarcinoma were cyclin D1 (CCND1), Cyclin Dependent Kinase Inhibitor 1A (CDKN1A) and E2F Transcription Factor 1 (E2F1; Fig. 2D).

GO enrichment and KEGG pathway analysis
The 114 interaction targets of Dioscoreae bulbiferae and Bruceae fructus and laryngocarcinoma were imported into the Metascape database for GO enrichment and KEGG pathway analysis.A total of 80 cellular components, 133 MFs, 374 BPs, and 89 KEGG pathways were obtained.The data was exported and sorted according to the P value.For each GO enrichment and KEGG pathway, 20 items with the lowest P value were selected to draw the figure.
The top 20 enrichment results of GO-MF were: enzyme binding, identical protein binding, protein binding, protein kinase binding, RNA polymerase II sequence-specific DNA binding transcription factor binding, protein homodimerization activity, transcription factor binding, transcription factor activity, sequence-specific DNA binding, protein kinase activity, transcription regulatory region sequence-specific  DNA binding, transcription coactivator binding, heme binding, RNA polymerase II transcription factor activity, ligand-activated sequence-specific DNA binding, ubiquitin protein ligase binding, sequence-specific DNA binding, protein serine/threonine kinase activity, protein heterodimerization activity, chromatin binding, steroid binding, kinase activity (Fig. 3B).
The top 20 enrichment results of GO-BP were: positive regulation of gene expression, negative regulation of apoptotic process, response to drug, positive regulation of transcription from RNA polymerase II promoter, positive regulation of transcription, DNA-templated, positive regulation of apoptotic process, positive regulation of cell proliferation, response to estradiol, cellular response to hypoxia, cellular response to cadmium ion,  apoptotic process, response to xenobiotic stimulus, response to lipopolysaccharide, cellular response to lipopolysaccharide, angiogenesis, extrinsic apoptotic signaling pathway in absence of ligand, positive regulation of pri-miRNA transcription from RNA polymerase II promoter, aging, positive regulation of angiogenesis, response to activity (Fig. 3C).
The top 20 KEGG enrichment results were: Pathways in cancer, Advanced Glycation End Product-Receptor for AGE signaling pathway in diabetic complications, Prostate cancer, Lipid and atherosclerosis, Hepatitis B, Bladder cancer, Fluid shear stress and atherosclerosis, Kaposi sarcoma-associated herpesvirus infection, Phosphoinositide 3-kinase-protein kinase B (PI3K-Akt) signaling pathway, Endocrine resistance, Pancreatic cancer, Chemical carcinogenesis-receptor activation, Hepatitis C, Human cytomegalovirus infection, Proteoglycans in cancer, interleukin-17 signaling pathway, Hepatocellular carcinoma, Cellular senescence, Small cell lung cancer, tumor necrosis factor (TNF) signaling pathway (Fig. 3D).Pathways in cancer are shown in the figure below (Fig. 4).

Construction of targets-pathways interaction network
The targets-pathways interaction network was constructed by Cytoscape software (Fig. 5A).

Molecular docking results
Molecular docking was carried out between the core components (diosbulbin B and brusatol) and the core targets (CCND1, CDKN1A, and E2F1).In general, when the binding free energy is <0, it indicates that the ligand and receptor can bind spontaneously. [22]The binding energy results obtained by molecular docking in this study were shown (Table 3).The molecular docking results with the lowest binding free energy were visualized by PyMOL.Diosbulbin B, and CDKN1A had low binding energy of −5.92 kcal/mol, indicating highly stable binding (Fig. 5B).Results showed that drug candidates bound to its protein targets through visible hydrogen bonds and strong electrostatic interactions.Moreover, the hydrophobic pockets of targets were occupied successfully by the candidate drugs.Cisplatin have been added as a control group, Cisplatin and CDKN1A had low binding energy of −7.273 kcal/mol.Cisplatin is a classic treatment for laryngocarcinoma, although the free energy of diosbulbin B is not as good as that of Cisplatin, it still has strong binding degree and effectiveness.

The results of vitro assays 3.7.1. Diosbulbin B inhibits the proliferation of human laryngocarcinoma cells.
To explore the biological effects of diosbulbin B, we first used the MTS technique to assess the vitality of laryngocarcinoma cells following diosbulbin B therapy.The results revealed that diosbulbin B suppressed Hep-2 cell activity in a concentration-dependent manner (Fig. 6A).Following that, we investigated the effects of diosbulbin B on colony formation after 14 days of culture of treated cells.Treatment with a low quantity of diosbulbin B somewhat reduced colony formation compared to the control (0 μg/mL diosbulbin B), but treatment with a high concentration of diosbulbin B greatly reduced colony formation (Fig. 6B).These findings revealed that diosbulbin B has powerful antiproliferative properties in Hep-2 cells.

Discussion
In the traditional Chinese medicine classic Compendium of Materia medica, Dioscorea fructus is mentioned that it has the effect of dissipating mass and detoxification.[25] Bruceae fructus has described in the traditional Chinese medicine classic Compendium of Materia medica that it has the effects of detoxification. [26]In modern pharmacological research, its extract Bruceae fructus oil has many advantages such as a wide anticancer spectrum, low toxicity, and good effect, and is widely used in the field of cancer. [27]n this study, we found that diosbulbin B and brusatol are the main core components of Dioscoreae bulbiferae and Bruceae fructus which are closely related to tumors. [28,29][32] Studies have shown that the knockdown of CircRNA CDR1as triggers low-dose DB (12.5 μM) -induced gastric cancer cell death, but has little effect on hepatocyte proliferation and apoptosis. [33]Studies have shown that brusatol is a promising anticancer compound that acts as a sensitizer when used in combination with other anticancer regimens by disrupting redox homeostasis. [34]n this study, the core targets CCND1, CDKN1A and E2F1 were obtained according to the PPI.Laryngeal squamous cell carcinoma (LSCC) is a common head and neck malignancy, and increasing evidence shows that high expression of cyclin D1 (CCND1) is a key regulator of the G1 phase of the cell cycle, associated with poor prognosis of chemotherapy resistance and some solid malignancies, and based on multivariate analysis, we also found that CCND1 level is an independent prognostic factor in Head and neck squamous cell carcinoma patients. [35]The study identified a novel mechanism of regulation of the protein CDKN1A (also known as p21) by the serine/threonine kinase complex mammalian target of rapamycin complex 1.Our results demonstrate that the mammalian target of rapamycin complex 1 substrate EIF4E-binding protein 1 in its non-phosphorylated state interacts with p21 and promotes p21 degradation.In addition, we demonstrate the prevalence of this mechanism in head and neck squamous cell carcinomas and show that it strongly and is significantly associates with improved disease-specific survival, providing evidence for its clinical relevance. [36]The study showed that the enforced expression of miR-1205 attenuates the migration, growth, and invasion of LSCC cells.E2F1 was validated as a target of miR-1205, while E2F1 binds to the miR-1205 promoter and transcriptionally represses miR-1205 expression.Overexpression of E2F1 partially reversed the inhibitory effect of miR-1205 on LSCC cells. [37]hrough KEGG enrichment analysis, it was found that the main pathways included Pathways in cancer, Advanced Glycation End Product-Receptor for AGE signaling pathway in diabetic complications, Prostate cancer, Lipid and atherosclerosis, Hepatitis B, Bladder cancer, Fluid shear stress and atherosclerosis, Kaposi sarcoma-associated herpesvirus infection, PI3K-Akt signaling pathway, Endocrine resistance, Pancreatic cancer, Chemical carcinogenesis -receptor activation, Hepatitis C, Human cytomegalovirus infection, Proteoglycans in cancer, interleukin-17 signaling pathway, Hepatocellular carcinoma, Cellular senescence, Small cell lung cancer, TNF signaling pathway.Among them, the PI3K-Akt signaling pathway plays a crucial role in regulating cell survival, growth, proliferation, angiogenesis, transcription, translation and metabolism. [38]PI3K-Akt is a unique master regulator of various cancers and can also regulate the occurrence and development of laryngeal cancer. [39]As a tumor promoter, the TNF signaling pathway plays an important role in regulating the occurrence and metastasis of tumors. [40]Studies have shown that by regulating the TNF signaling pathway, Protein Tyrosine Phosphatase Non-Receptor Type 2 is highly expressed in laryngeal cancer and can regulate the proliferation of laryngeal cancer. [41]According to the pathway enrichment analysis, the pathways enriched by the target genes of the effective components of Dioscorea bulbifera and Brucea javanica mainly affect the occurrence, development, and prognosis of laryngeal cancer by regulating cell growth, cell proliferation, angiogenesis and tumor occurrence and metastasis.Diosbulbin B and CDKN1A had low binding energy of −5.92 kcal/mol, indicating highly stable binding.Results showed that drug candidates bound to its protein targets through visible hydrogen bonds and strong electrostatic interactions.To investigate the biological effects of diosbulbin B, we assessed the viability of laryngocarcinoma cells after diosbulbin B therapy using the MTS technique.The findings demonstrated that diosbulbin B inhibited Hep-2 cell activity in a concentration-dependent manner.Furthermore, in Hep-2 cells, diosbulbin B shows potent antiproliferative effects.Flow cytometry was utilized to detect apoptosis in order to examine the influence of diosbulbin B on laryngocarcinoma cell apoptosis.The findings revealed that diosbulbin B induced apoptosis in laryngocarcinoma cells in a concentration-dependent way.

Conclusions
Based on network pharmacological analysis, this study demonstrated that Dioscoreae bulbiferae and Bruceae fructus treated laryngocarcinoma through multi-compounds, multi-targets, and multi-pathways, and preliminarily clarified the related potential mechanism of Dioscoreae bulbiferae and Bruceae fructus in the treatment of tumor.Through KEGG pathway enrichment analysis, it was found that Dioscoreae bulbiferae and Bruceae fructus played an important role in the treatment of laryngocarcinoma, including PI3K-Akt signaling pathway, TNF signaling pathway, and so on.These pathways mainly affect the development and prognosis of laryngeal cancer by regulating cell growth, cell proliferation, angiogenesis, and tumorigenesis and metastasis.The molecular docking results showed that the affinity between core components and core targets was good.The vitro assays demonstrated that diosbulbin B inhibited Hep-2 cell activity in a concentration-dependent manner.Furthermore, in Hep-2 cells, diosbulbin B shows potent antiproliferative effects.Flow cytometry results revealed that diosbulbin B increased apoptosis in laryngocarcinoma cells in a concentration-dependent manner.Although subsequent validations are needed to determine the exact mechanism of Dioscoreae bulbiferae and Bruceae fructus, our present study provides promising directions for future research.

Figure 3 .
Figure 3. (A) The top 20 enrichment results of GO-CC.(B) The top 20 enrichment results of GO-MF.(C) The top 20 enrichment results of GO-BP.(D) The top 20 enrichment results of KEGG.BP = biological processes, GO = gene ontology, KEGG = Kyoto encyclopedia of genes and genomes, MF = molecular function.

Table 2
Active components of Bruceae fructus.

Table 3
Molecular docking results.